Nucleic acids encoding peptides for treating wounds, anti-angiogenic compounds and uses thereof

ABSTRACT

The present disclosure relates to protein and peptide chemistry. More particularly, it relates to compounds, compositions and uses thereof for promoting and inhibiting angiogenesis. The peptides of the present disclosure include peptides comprising SEQ ID NOs: 1-4 which promote angiogenesis and cell proliferation. Further, the anti-angiogenic compounds of the present disclosure include antisense oligonucleotides that hybridize or are complementary to the polynucleotides of SEQ ID NOs: 5-16, and the like.

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application Ser. No. 61/197,420 filed on Oct. 27,2008. The contents of the provisional application are herebyincorporated by reference in their entirety.

TECHNICAL FIELD

The present disclosure relates to protein and peptide chemistry. Moreparticularly, it relates to compounds, compositions and uses thereof forpromoting angiogenesis for wound healing and other aspects ofregenerative medicine and inhibiting pathologic angiogenesis causingarthritis, complicating or compromising sight during aging (maculardegeneration), diabetes (proliferative diabetic retinopathy) andresponsible for tumor growth and metastasis.

BACKGROUND

It is widely recognized that the human body's response to injury iscomplex, and is dependent on a panoply of signaling pathways expressedby several cell and tissue types over an extended period of time.Importantly, the wound “bed” undergoes significant remodeling asre-epithelialization ensues concomitant with dermal angiogenesis. And,while wound healing typically occurs as a natural, uneventful processleaving the individual with neither noticeable scars nor wounds thatchronically persist, this is not the case for 2-3% of the U.S.population. For these individuals, excessive scarring and chronic woundsare sustained as medical issues requiring specialized treatment,individualized care, or in some cases, hospitalization. Thus, whileacute wound healing may occur in a matter of days or weeks, chronicwounds can remain in an open state for months and even years.

Under normal circumstances, the process of human wound healing can bebroken down into three phases. An initial inflammatory phase, which isfollowed by robust tissue remodeling and proliferation (theproliferative phase), is ultimately succeeded by a “maturational phase”in which reepithelialization, dermal angiogenesis, and wound closureensue. The inflammatory phase is characterized by homeostasis, with aprovisional matrix contributed by the blood itself creating the initialwound bed. As basement membrane and interstitial collagens are exposedduring injury, blood platelets are stimulated to release multiplechemokines, including epidermal growth factor (EGF), fibronectin,fibrinogen, histamine, platelet-derived growth factor (PDGF), serotonin,and von Willebrand factor, to name several. These factors help tostabilize the wound through clot formation and control bleeding, thereinlimiting the extent of injury. Platelet degranulation also initiates thecomplement cascade, specifically via C5a, which is a potentchemoattractant for neutrophils.

The timeline for cell migration in a normal wound-healing process isalso well ordered, with an inflammatory phase beckoning the migration ofimmune response cells. For example, neutrophils function todecontaminate the wound from foreign debris via phagocytosis withsupport from immigrating macrophages. In turn, macrophages releasecytokines that locally function to stimulate a robust proliferativeresponse required for tissue morphogenesis and healing. It is during theproliferative phase that re-epithelialization and angiogenesispredominate. The entire process represents a dynamic and reciprocalcontinuum, with the angiogenesis of wound healing propagating andsustaining the re-epithelialization and tissue remodeling processes: allevents continued until the wound site reaches maximal strength, perhapsas long as 1 year post-injury.

Throughout life, the vasculature undergoes significant morphogenesis.Two independent but related processes govern the formation of the adultvasculature: vasculogenesis and angiogenesis. Initially duringvasculogenesis, immature vessels are formed de novo from endothelialcell precursors, the angioblasts, which proliferate and coalesce,creating a capillary plexus. Local differentiation of endothelial cellsserves as an initiating event for the subsequent rounds of vascular“budding” or “sprouting,” angiogenesis, which gives rise to the systemof arteries, veins, arterioles, venules, and capillaries. Interestingly,in the adult, physiologic angiogenesis occurs during the femalereproductive cycle, but otherwise the pre-dominant form of physiologicangiogenesis during adult life occurs during wound healing.

Many positively and negatively acting factors influence the angiogenesisof wound healing, including the microenvironment in which vascularmorphogenesis occurs. Soluble polypeptides, cell-cell and cell-matrixinteractions, and hemodynamic and biomechanical forces all playstrategic roles. More recently, we have learned that blood vesselsprouting during wound healing is likely to be critically dependent on awell-ordered signaling cascade responsible for regulating microvascularcytoskeletal function. In addition, clear-cut roles for theextracellular matrix and the repertoire of metalloproteinasescontrolling matrix remodeling also play modulatory roles in fosteringwound-healing angiogenesis.

Investigations and procedures for examining and measuring healing,migration, and formation of new blood vessels that occur during theresponse to injury, and which are impaired during chronic wound healingduring diabetes, venous stasis ulceration, pressure ulcer formation andischemia-reperfusion disorders: all, are known in the art. See, forexample, Demidova-Rice et al., Lasers in Surgery and Medicine 39:706-715(2007); Kutcher et al., Am. J. Pathol. 171:693-701 (2007); Herman, etal., Stewart Martin, ed. (2007); Herman, D. Shepro, ed. Elsevier Pub,Inc. (2006); Riley et al., J. Burns & Wounds 4:141-59, (2005); Papettiet al., Am. J. Physiol 282:947-970, (2002); Papetti et al., Am. J.Pathol. 159:165-77 (2001); the disclosures of which are all incorporatedherein by reference.

There is, therefore, a great interest and need in developingcompositions and devices that are useful for treating wounds. See, forexample, Gandy, U.S. Patent Publication No. 20080213238; Gandy U.S.Patent Publication No. 20060142198; Gandy U.S. Patent Publication No.20060004189; Gandy U.S. Patent Publication No. 20050191286; and GandyU.S. Patent Publication No. 20040197319, the disclosures of which areall incorporated herein by reference.

Other attempts to prepare wound healing compositions are disclosed in,for example, Knighton, U.S. Pat. Nos. 5,165,938 and 4,957,742, whichdisclose platelet enriched plasma produced from blood wherein theplatelets are activated by thrombin which causes the release ofplatelet-derived growth and angiogenesis factors. A carrier, such as amicrocrystalline collagen, is added to produce a wound-treating salve,and the composition is applied directly to wounds and initiates healingin non-healing wounds as well as accelerating normal wound-healing byincreasing vascularization, stimulating fibroblast mitosis andmigration, and increasing collagen synthesis by fibroblasts. It is saidthat the composition may also be applied to tissue to facilitate thegrowth of hair.

Worden, U.S. Pat. No. 6,524,568, discloses a platelet gel wound healingcomposition that includes growth factors and ascorbic acid andoptionally including an antioxidant such as Vitamin A and/or Vitamin E.Antibiotics may also be included. Chao, U.S. Pat. No. 5,185,160,discloses a heat-treated, viral-inactivated platelet membranemicroparticle fraction which may be prepared from outdated platelets.The microparticle fraction is said to be substantially free of plateletghosts and may be used as a pharmaceutical preparation in transfusions.Chao, U.S. Pat. No. 5,332,578, also discloses a heat-treated,viral-inactivated platelet membrane microparticle product which may beprepared from outdated mammalian platelets. The microparticle product issaid to contain isolated platelet membrane fragments that are free ofalloantigens and GP IIb/IIIa complexes and it is said that the productmay be used as a pharmaceutical preparation in transfusions.

Crowe, U.S. Patent Publication No. U.S. 2004/0265293A1, discloses adehydrated composition that includes freeze-dried platelets. Theplatelets are loaded with trehalose in an amount from about 10 mM toabout 50 mM, and at a temperature of from greater than about 25° C. toless than about 40° C. The freeze-dried platelets are said to besubstantially shelf-stable and are rehydratable so as to have a normalresponse to an agonist, for example, thrombin, and it is said thatvirtually all of the platelets participate in clot formation withinabout three minutes at 37° C.

Van der Meulen et al., J. Membrane Biol. 71:47-59 (1983), disclosesporcine alpha-granules that were found to be essentially homogeneous bytransmission electron microscopy. Freeze-fractured samples of isolatedgranules revealed intramembranous particles on the exoplasmic fracturesurface and, to a lesser extent, on the protoplasmic fracture surface,whereas the PS (protoplasmic) surface was relatively smooth and, it issaid, the granules appeared to be sealed. Membranes were isolated byalkali extraction of the granules which removed protein andphospholipids yielding membrane vesicles devoid of the dense core. Themembranes were said to contain major and minor polypeptides. Theexposure of specific proteins on the cytoplasmic surface of the granulemembrane was also determined. In sealed granules, bands were modified bythe reagents, and a fraction eluted by alkali extraction was alsoanalyzed and found to contain nine major polypeptides.

Chao et al., Transfusion 36:536-542 (1996), discloses preparation of IPMfrom outdated platelets. The platelets were disrupted by freezing andthawing, washed and heated to inactivate possible viral contaminants,and then a sonicated membrane microvesicle fraction was separated andlyophilized. The hemostatic activity of IPM was measured by its abilityto reduce the prolonged bleeding time in thrombocytopenic rabbits.According to Chao, administration of IPM at a dose of 2 mg per kgresults in a substantial reduction in the bleeding time. It is reportedthat, in a series of 23 experiments, a median preinjection bleeding timeof 15 minutes was reduced to 6 minutes within 4 hours after IPMadministration. Administration of IPM was said to show a mildenhancement in the thrombogenicity index, as measured in the Wesslerrabbit model, which was not significant. Chao concludes that IPM mayhave clinical potential as a substitute for platelets in the treatmentof bleeding due to thrombocytopenia.

Gogstad, Thrombosis Research 20:669-681 (1980), discloses a method forthe isolation of alpha-granules wherein a two-step French pressure cellhomogenization procedure produced an organelle concentrate for loadingon density gradients. The procedure was said to be optimalized withrespect to recovery of intact alpha-granules. The organelle homogenatewas loaded to 17.5-27.5% metrizamide gradients and centrifuged.Organelle aggregate formation was said to be minimized by controllingthe ionic conditions and the shape of the gradient. The alpha-granuleswere separated from lysosomes and dense bodies, but overlapped with themitochondria, and the alpha-granules were recovered from the gradient toomit the major amount of mitochondria from the final preparation.

Hernandez, Vox Sang 73:36-42 (1997), discloses an investigation into theeffects on hemostatsis of nonliving platelet derivatives. The effects ofdifferent platelet preparations on primary hemostatsis in awell-established perfusion model were evaluated, and studies werecarried out with blood anticoagulated with low molecular weight heparin.Frozen-thawed, sonicated or lyophilized platelets were added to normalblood or to blood which had been experimentally depleted of platelets.Platelet interaction with the subendothelium and fibrin deposition weremorphometrically evaluated. Hernandez reports that addition ofnon-viable platelet preparations to thrombocytopenic blood promoted astatistically significant increase in the deposition of fibrin on thesubendothelium, but only lyophilized platelets retained some ability tointeract with the subendothelium. Flow cytometry studies demonstratedthe presence of GPIb, GPIIa and P-selection on lyophilized platelets.Hernandez concludes that preparations containing non-viable plateletsmay still retain some hemostatic properties.

Blood vessels are the method by which oxygen and nutrients arecirculated and supplied to tissue, as well as the method by which wasteproducts are removed from such tissue. Angiogenesis refers to theprocess by which new blood vessels are formed from preexisting bloodvessels. See, for example, the review by Folkman and Shing, J. Biol.Chem. 267:10931-10934 (1992), Dvorak et al., J. Exp. Med. 174:1275-1278(1991). Accordingly, angiogenesis is generally considered an essentialbiological process, which includes instances where a greater degree ofangiogenesis is desired, such as wound healing, as discussed above.However, abnormal or inappropriate angiogenesis, where there isexcessive blood vessel proliferation, can lead to severe negativeoutcomes, as exemplified in vascularized ocular diseases such asproliferative diabetic retinopathy, and wet age-related maculardegeneration and in cancers, where solid tumor growth has beendemonstrated to be angiogenesis-dependent, with the newly developed orangiogenic microvessels transforming dormant avascular micrometastasesinto actively growing macroscopic tumors, which derive an ample supplyof oxygen and nutrients from angiogenic tumor microvessels.

It is known that tumor growth impacts a large number of people eachyear, e.g., Cancer accounts for 7.1 million deaths annually (12.5% ofthe global total). Approximately 20 million people suffer from cancer; afigure projected to rise to 30 million within 20 years. The number ofnew cases annually is estimated to rise from 10 million to 15 million by2020 (World Health Organization).

With respect to angiogenesis-dependent visually blinding disordersaccompanying aging and diabetes, there is a comparably staggering andever-increasing number of affected individuals. For example, withrespect to visually blinding disorders observed during aging, angiogenicor vascular complications of AMD account for roughly 10 percent of allthose patients suffering with AMD but, “wet” AMD accounts for 90 percentof all AMD-associated blindness: roughly 2.3 million of the 34 millionAmericans over age 70 will be affected. And, with respect to thevisually blinding vascular complications associated with diabetes, it islikely that between 40 to 45 percent of Americans diagnosed withdiabetes have some stage of diabetic retinopathy (www.nei.nih.gov).

Cancer is becoming an increasingly important factor in the global burdenof disease. The estimated number of new cases annually is expected torise from 10 million in 2000 to 15 million by 2020. Some 60% of thesecases will occur in the less developed parts of the world. More than 7million people now die each year from cancer. Yet with the existingknowledge, at least one-third of cancer cases that occur annuallythroughout the world could be prevented. The use of therapies designedto inhibit angiogenesis or neovascularization may significantly effectthe growth of solid tumors and development of ocular disease. Throughblocking angiogensis or neovascularization, tumor growth and oculardisease can be inhibited suggesting that these diseases require thecontinued blood vessel growth for progression of the tumors or oculardisease. Inhibition of angiogenesis or neovascularization is, therefore,a promising anti-cancer treatment and an anti-ocular angiogenictherapeutic approach as has recently been demonstrated (anti-VEGF).

Inhibition of angiogenesis may also be useful in treating diseases thatare characterized by unregulated blood vessel development including, forexample, vascular tumors (e.g., sarcomas, carcinomas, and lymphomas) andocular diseases (e.g., macular degeneration and diabetic retinopathy).Cancer cells, as used herein, include tumors, tissue, and the like.

As can be seen from the above, there is a great need for and interest indeveloping compounds and compositions that are useful in treating andhealing wounds, such as chronic wounds caused by diabetes which aredifficult to heal. Not all current methods of treating chronic woundshave been successful. It is therefore an object of the presentdisclosure to identify new compounds and methods that will promote andimprove wound healing, especially for chronic and otherwise unhealablewounds. Compounds and compositions that specifically promotekeratinocyte migration to a wound, and which may not promote substantialkeratinocyte proliferation, on the one hand, and which enhanceendothelial cell formation, and which may not promote substantialendothelial cell migration or proliferation, would represent asignificant advance over the products that are currently available forhealing wounds. Further, there is great need for and interest indeveloping compounds and compositions that are useful in combatingvascular tumors and certain vision-threatening complications resultingfrom abnormal angiogenesis, such as, for example, diabetic retinopathyor age-related macular degeneration. It is therefore another object ofthe present disclosure to identify novel compounds and compositions thatinhibit abnormal angiogenesis, but do not ablate the complement ofphysiologic blood vessels or survival of normal cells and tissues thatare required for normal organismic functionality. Disclosure of suchapproaches and/or entities would represent a significant advance overthe current state of the art, standard of care or products that arecurrently available or are in use.

SUMMARY

In one object of the present disclosure, molecules or compounds andcompositions for promoting angiogenesis through stimulation and growthof tissues and vascularization are provided. More particularly, themolecules, compounds, and compositions include the wound healingpeptides and nucleic acid sequences described herein, which are isolatedand purified or substantially purified. The molecules, compounds, andcompositions comprising the wound healing molecules or compounds, may beused to treat insults to the body, such as burns, cuts, and scrapes;contusions, including oral and otolaryngological wounds; wounds that arecaused and treated by plastic surgery; and bone damage.

Another object of the disclosure is directed to the wound healingpeptides of SEQ ID NOs:1-4 and combinations thereof. One object of thedisclosure relates to the inventive composition comprising an effectiveamount of any one of or combination of the wound healing compounds, suchas for example peptides of SEQ ID NOs: 1-4 and combinations thereof,described herein with a pharmaceutically- or physiologically-acceptablecarrier, vehicle, or diluent. A therapeutic amount of the wound healingpeptide of the disclosure is administered either directly or in acomposition to a subject having a wound, including, for example, anacute accidental injury, surgical acute injuries, chronic wounds,ulcers, thermal injuries, combat casualty injuries, or a subject proneto hypertrophic scarring, such as keloid scarring. As used herein,“subject” refers to a mammal, including humans, horses, canines,felines, and the like.

In a further object, these wound healing compounds and compositions maybe used alone or in combination therapy together with other growthpromoting actives, such as isolated and purified or syntheticallyproduced growth factors, and/or pain and inflammation reducing factors.

Another object of the present disclosure provides methods of using thewound healing compounds and compositions prepared, alone or incombination therapy. These wound healing compounds and compositions maybe administered to a subject in need thereof in an amount effective totreat and/or heal a wound. Thus, the present disclosure also providesmethods of treating subjects by administering to a subject in needthereof an effective amount of the wound healing compound or compositionaccording to the disclosure as described herein. The present disclosureprovides a particularly useful method of treating a patient sufferingfrom diabetes or another disorder that prevents normal wound healing, ora subject being treated with a medication that prevents normal woundhealing, by administering to the subject in need thereof an effectiveamount of a molecule or composition according to the instant disclosure.

A further object of the disclosure provides anti-angiogenic compounds,such as oligonucleotide sequences (also known as antisenseoligonucleotides) that hybridize to or are complementary to thenucleotide sequences of SEQ ID NOs:5-16 for treating a disease,disorder, or condition associated with abnormal neovascularization orangiogenesis.

One object of the disclosure relates to the inventive compositioncomprising an effective amount of any one of or combination of theanti-angiogenic compounds described herein (including antisenseoligonucleotides of the disclosure) with a pharmaceutically- orphysiologically-acceptable carrier, vehicle, or diluent.

A further object of the present disclosure provides a method forinhibiting angiogenesis, tumor growth and metastasis in the tissue of asubject or mammal in need thereof, by administering to the subject ormammal, an anti-angiogenic compound or composition comprising theanti-angiogenic compound, including for example, antisenseoligonucleotides to any of the disclosed nucleotides, as describedherein.

Further, in the methods of the present disclosure, a therapeutic amountof an anti-angiogenic compounds of the invention is administered to ahuman or other mammal having a disease or disorder/condition associatedwith abnormal neovascularization or angiogenesis, as described above.

Another object of the disclosure is a kit that includes elements forpreparing and/or administering a wound treating molecule or composition.The kit includes a wound healing molecule or composition contained in aform to be applied to a wound, an applicator element for applying thewound healing molecule or composition to a wound, and optionallyadditional devices or materials useful for the preparation of the woundor administration of the wound healing molecule or composition.

Yet another object of the disclosure is a kit that includes elements forpreparing and/or administering an anti-angiogenic compound of thepresent disclosure. The kit includes an anti-angiogenic compoundcontained in a form to be administered to a subject, and optionallyadditional devices or materials useful for administration of theanti-angiogenic compound.

These and other aspects of the present disclosure will become apparentto those skilled in the art after a reading of the following detaileddescription, including the illustrative embodiments and examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of a comparison 0.5 nM of the peptide of SEQID NO: 1 (“UN1”) to a serum control sample in the formation of tubulesfrom Human Microvascular Endothelial Cells (HMVEC) in Matrigel™ after 3hours. The number and lengths of the processes or tubules formed afterthe addition of 0.5 nM of the peptide of SEQ ID NO: 1 shows significantinduction of in vitro angiogenesis compared with the control.

FIG. 2 is a graph illustrating a measure of angiogenesis in terms oftotal process length in Human Microvascular Endothelial Cells (HMVEC)with 0.4 nM and 250 nM of the SEQ ID NO: 1 (“UN1”).

FIG. 3 is a graph illustrating a measure of angiogenesis in terms oftotal process length in Human Microvascular Endothelial Cells (HMVEC)with 0.4 nM and 250 nM of the peptide of SEQ ID NO: 3 (“UN3”).

FIG. 4 is a graph illustrating a measure of relative wound closure viaNormal Human Epithelial Keratinocyte (NHEK) migration after exposure to0.5 nM of each of the peptides of SEQ ID NOs: 1, 2, and 3 (“UN1,” “UN2,”and “UN3” respectively). The relative wound closure was significantlybetter for each of the peptides compared to “control.”

DETAILED DESCRIPTION Peptides and Nucleic Acids

Embodiments of the instant disclosure include compounds, compositionsand uses thereof for promoting and inhibiting angiogenesis. These woundhealing and anti-angiogenic compounds may be used in the treatment ofwounds and abnormal vascular diseases, including for example, cancers.Anti-angiogenic compounds encompass compounds used foranti-vascularization, anti-proliferation, and the like. Additionally,aspects of the instant disclosure are useful for diagnostic assays.

One embodiment of the disclosure provides isolated or purified peptidesof SEQ ID NOs: 1-4 as wound healing compounds. As is known, peptides areformed by linking of amino acids through peptide bonds. Peptides aremade up of only a few amino acids; whereas proteins generally havegreater than 50 amino acid residues.

The peptides of the disclosure may be prepared in any suitable manner.Such peptides include isolated naturally occurring peptides,recombinantly produced peptides, synthetically produced peptides, orpeptides produced by a combination of these methods. The means forpreparing such peptides are well understood in the art. The peptides maybe a part of a larger protein, such as a fusion protein. The inclusionof additional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification, such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production is also advantageous.

A further embodiment of the disclosure relates to peptides that are inan isolated form and substantially purified. A recombinantly producedversion of a peptide can be substantially purified using techniquesdescribed herein or otherwise known in the art, such as, but not limitedto, for example, by Affinity Chromatography, Ion ExchangeChromatography, Hydrophobic Interaction Chromatography, Gel Filtration,Reversed Phase Chromatography, and the like. Peptides of the inventionalso can be purified from natural, synthetic or recombinant sourcesknown in the art.

In another embodiment, peptides of the present disclosure are composedof amino acids joined to each other by peptide bonds or modified peptidebonds, i.e., peptide isosteres, and may contain amino acids other thanthe 20 gene-encoded amino acids. The peptides may be modified by eithernatural processes, such as posttranslational processing, or by chemicalmodification techniques which are well known in the art. Suchmodifications are detailed in basic texts, as well as in the vastresearch literature.

Modifications can occur anywhere in a peptide, including the peptidebackbone, the amino acid side-chains and the amino or carboxyl termini.It will be appreciated that the same type of modification may be presentin the same or varying degrees at several sites in a given peptide. Anygiven peptide may contain multiple types of modifications. For example,the peptides may be branched, as a result of ubiquitination, and theymay be cyclic, with or without branching. Cyclic, branched, and branchedcyclic peptides may result from natural posttranslational processes ormay be made synthetically. Non-limiting modifications includeacetylation, acylation, ADP-ribosylation, amidation, covalent attachmentof flavin, covalent attachment of a heme moiety, covalent attachment ofa nucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent cross-links, formation of cysteine, formation ofpyroglutamate, formylation, gamma-carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, pegylation, proteolytic processing,phosphorylation, prenylation, racemization, selenoylation, sulfation,transfer-RNA mediated addition of amino acids to proteins such asarginylation, and ubiquitination. (See, for instance, Structure inProtein Chemistry, 2^(nd) Ed. Jack Kyte, Taylor & Francis, Inc., NewYork (2006); Proteins—Structure and Molecular Properties, 2^(nd) Ed., T.E. Creighton, W. H. Freeman and Company, New York (1993);Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pgs. 1-12 (1983).

The peptides may be combined in different combinations and orders. Forexample, the peptides of SEQ ID NOs: 1 and 2 are linked, from N-terminusto C-terminus, with a linker, for example, proline (“Pro”) to create SEQID NO: 3. The two peptides can be linked with any amino acid orartificial synthetic molecule that would enable attachment of the twopeptides. Another embodiment is the peptides of SEQ ID NOs: 1 and 2 arelinked, from N-terminus to C-terminus, with a linker, for example,proline (“Pro”) to create SEQ ID NO: 4. Peptides of the disclosureencompass any of the peptides, in any order, in any combination. Forexample, an embodiment of the present disclosure could be: [SEQ ID NO:A]_(m)—X—[SEQ ID NO: B]_(n), where “A” and “B” are different sequencesdisclosed herein, “X” is a linker (e.g., proline), and “m” and “n” areany positive integer including zero except that m and n cannot besimultaneously be zero.

The compounds of the present disclosure may be isolated and purified bytechniques known in the art. Example 1 describes the isolation andidentification of the inventive peptides. Techniques include, such as,for example, by solubility, size, charge, hydrophobicity, and byaffinity. The wound healing and anti-angiogenic compounds used incompositions of the disclosure not only comprise of a pharmaceutically-or physiologically-acceptable carrier, but also optionally, any othercomponent that may seem fit for the benefit of the intended purpose ofwound healing.

A further embodiment of the disclosure provides a polynucleotidecomprising, or alternatively consisting essentially of or consisting of,the sequence identified in or a polynucleotide of any one of SEQ ID NOs:5-16. Yet another embodiment relates to polynucleotides encoding thepeptides of the disclosure comprising, or alternatively consistingessentially of or consisting of, the peptide sequences of SEQ IDNOs:1-4. The polynucleotide sequence of SEQ ID NOs: 5, 9, and 13 encodeeach of the amino acid sequences or peptides of SEQ ID NOs: 1, 2, and 3,respectively. The polynucleotide sequences can be synthesized in anumber of different ways, such as for example, reverse translation.Furthermore, where wobble nucleotide positions are possible, multiplenucleotides capable of encoding each peptide/protein are provided. Thus,for the amino acid sequence of SEQ ID NO: 1, the correspondingnucleotide sequences are SEQ ID NOs: 5-8. For the amino acid sequence ofSEQ ID NO: 2, the corresponding nucleotide sequences are SEQ ID NOs:9-12. For the amino acid sequence of SEQ ID NO: 3, the correspondingnucleotide sequences are SEQ ID NOs: 13-16.

LIST OF SEQ ID SEQUENCES SEQ ID NO: 1 GluLeuLeuGluSerTyrIleAspArgSEQ ID NO: 2 ThrAlaThrSerGluTyrGlnThrPhePheAsn ProArg SEQ ID NO: 3GluLeuLeuGluSerTyrIleAspArgProThr AlaThrSerGluTyrGlnThrPhePheAsnPro ArgSEQ ID NO: 4 ThrAlaThrSerGluTyrGlnThrPhePheAsnProArgProGluLeuLeuGluSerTyrIleAsp Arg SEQ ID NO: 5GAACTTCTTGAATCTTATATTGATCGT SEQ ID NO: 6 GAGTTCTTCGAGAGCTACATCGACAGCSEQ ID NO: 7 GAACTACTAGAATCATATATAGATCGA SEQ ID NO: 8GAACTGCTGGAATCGTATATTGATCGG SEQ ID NO: 9ACTGCTACTTCTGAATATCAAACTTTTTTTAA TCCTCGT SEQ ID NO: 10ACCGCCACCAGCGAGTACCAGACCTTCTTCAA CCCCAGC SEQ ID NO: 11ACAGCAACATCAGAATATCAAACATTTTTTAA TCCACGA SEQ ID NO: 12ACGGCGACGTCGGAATATCAAACGTTTTTTAA TCCGCGG SEQ ID NO: 13GAACTTCTTGAATCTTATATTGATCGTCCTAC TGCTACTTCTGAATATCAAACTTTTTTTAATC CTCGTSEQ ID NO: 14 GAGTTCTTCGAGAGCTACATCGACAGCCCCACCGCCACCAGCGAGTACCAGACCTTCTTCAACC CCAGC SEQ ID NO: 15GAACTACTAGAATCATATATAGATCGACCAAC AGCAACATCAGAATATCAAACATTTTTTAATC CACGASEQ ID NO: 16 GAACTGCTGGAATCGTATATTGATCGGCCGACGGCGACGTCGGAATATCAAACGTTTTTTAATC CGCGG

In another embodiment, the polynucleotides of the present disclosure maybe composed of any polyribonucleotide or polydeoxyribonucleotide,including modified RNA or DNA or unmodified RNA or DNA. For example,polynucleotides may comprise single- and double-stranded DNA, single-and double-stranded RNA, DNA that is a mixture of single- anddouble-stranded regions, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded or a mixtureof single- and double-stranded regions. Additionally contemplated arepolynucleotide that are comprised of triple-stranded regions or RNA orDNA or both RNA and DNA. A polynucleotide may also contain one or moremodified bases or DNA or RNA backbones modified for stability or forother reasons. Modified bases include, for example, methylated ortritylated bases and unusual bases such as inosine. DNA and RNA may bemade modified in a variety of ways. Hence, polynucleotides of theinstant disclosure also embrace chemically, enzymatically, ormetabolically modified forms.

DNA shuffling, which includes but is not limited to, gene-shuffling,motif-shuffling, exon-shuffling, and codon-shuffling, are techniquesuseful in generating fusion proteins of the disclosure. The activitiesof peptides and polypeptides of the disclosure may be modulated oraltered by using the DNA shuffling technique to generate agonists orantagonists of the peptides or polypeptides. See, for example, U.S. Pat.Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, andPatten et al., Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama,Trends Biotechnol. 16(2):76-82 (1998); Hansson, et al., J. Mol. Biol.287:265-76 (1999); and Lorenzo and Blasco, Biotechniques 24(2):308-13(1998).

In one embodiment, the inventive polynucleotides disclosed herein thatcorrespond to SEQ ID NOs: 5-16 and the peptides encoded by thesepolynucleotides may be attained by DNA shuffling. The assemblage of twoor more DNA segments by homologous or site-specific recombination maygenerate variation in the polynucleotide sequence by DNA shuffling.Another embodiment relates to the polynucleotides of the invention, orthe encoded peptide or polypeptides, that may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of a polynucleotide encoding a peptide or polypeptideof the invention may be recombined with one or more components, motifs,sections, parts, domains, fragments, etc. of one or more heterologousmolecules.

Formation of an Expression Vector

An expression vector of the present disclosure is a vector containing atleast one of the polynucleotides of SEQ ID NOs: 5-16 as described above.A DNA vector of the present invention may be prepared by ligating aplasmid DNA having an artificial nucleotide sequence to the DNA moleculeof the present invention. For example, the DNA of interest may beinserted into the vector by using two different restriction enzymes forcutting both the vector and insert, and then ligating the ends. However,when the ends are blunt ends, a linker may be utilized to facilitate theligation and insertion of the DNA of interest. The linker is at leastone or two more nucleotide(s) that is/are not naturally connected to theDNA molecule of the present invention, and is appropriately designeddepending on the site of the vector to be inserted.

Non-limiting examples of expression vectors, including DNA vectors inwhich the DNA molecule of the present invention is integrated, include aplasmid such as pBR322, pBR325, pUC7, pUC8, pUC18, pUC19, pBluescript orpGEM; a cosmid such as pHC79; and a phage such as pUC19, or M13 phage.The vector is digested with appropriate restriction enzymes and the DNAmolecule disclosed herein with or without additional sequence, such aslinker, is inserted therein by standard molecular procedure known andunderstood by the scientist.

A DNA vector to construct the expression vector for a polynucleotideencoding peptide of Sequence ID Nos. 1-3 is not limited, and may bechosen from any of the aforesaid vectors or those known in the art. ADNA vector to express the objective protein as a fusion protein with aTag, by which the objective protein could be affinity-purified, is moresuitable. For examples of such a vector, pGEX vector (AMERSHAMBIOSCIENCES Corp.) or pQE vector (QIAGEN Inc.) is commerciallyavailable.

The transcriptional regulation region, comprising a promoter and aterminator, is necessary for inclusion in the expression vector. Thesuitable promoter differs in host-by-host cell. For example, lac, tac orT5 promoter is used as promoter in the case of E. coli as host cell.OAX1 or GAPDH promoter in the case of yeast, polyhedrin promoter in thecase of insect cell, or CMV or β-actin promoter in the mammalian cellsare suitable.

Host Cell

Using the resulting expression vector as described above, a variety ofhost cells can be appropriately transformed to obtain a microorganism orcells capable of producing the peptides of SEQ ID NOs: 1-4 or arecombinant protein comprising a part of any of SEQ ID NOs: 1-4 and aTag sequence. The appropriate transformation methods are well known toone skilled in the art.

Host cells used herein can be selected in terms of compatibility of theexpression vector, suitability of the products, etc., and may be eitherprokaryotic or eukaryotic cells. Specific examples of host cellsinclude, but are not limited to, bacteria such as the genus Escherichia(e.g., E. coli) or the genus Salmonella (e.g., Salmonella typhimurium),and lower eukaryotic cells such as yeast (e.g., Saccharomycescerevisiae) or fungi (e.g., Penicillium). Non-limiting examples of hostcells in higher eukaryotic cells are insect cells, Chinese hamster ovary(CHO) cells, CEF cells, or human cell lines (e.g., HeLa).

Expression of the Wound Healing Peptide

Host cells transformed with an appropriate expression vector can becultured and proliferated under incubation conditions well known to oneskilled in the art.

For example, the transformed E. coli can be well grown in LB medium at37° C., under aerobic conditions. In producing a peptide of the presentinvention, the condition for the induction of the protein can be chosenaccording to the used promoter. In the case of E. coli lactose promoterand operator system, as a specific example, it is achieved by adding anappropriate amount of isopropyl-1-thio-β-D-galactopyranoside (IPTG) to aculture medium.

A method to purify the peptides of the invention is not particularlylimited, and any known method is applicable to the purification incombination with techniques well known in this field. When the peptidesof the invention are expressed as a fusion protein containing some Tagthat can be used in purification through an affinity column, theaffinity column is a very convenient tool. For example, the peptides ofthe invention expressed as fusion with glutathione S-transferase (GST)using pGEX vector could be purified easily through Glutathione Sepharose4B column (AMERSHAM BIOSCIENCES Corp). Thus, in another embodiment, thepresent disclosure includes a prokaryotic or eukaryotic host celltransformed or transfected with a DNA sequence as described above in amanner allowing the host cell to express the polypeptide.

Viral Transduction In Situ—Peptide Adenovirus Construction

The coding sequence for the peptides of the disclosure can be clonedinto an appropriate vector, such as a pEF-BOS vector, into, for example,pAdTrack-CMV, to take advantage of a forward primer containingrestriction sites that would enable tagging the peptide with adefinitive expression tag, if needed, e.g., a KpnI site, a Kozakconsensus site, and a region targeting a myc sequence(5′GCGGTTACCACCACCATGGAACAAAAACTCATCTCAGAA-3′) (SEQ. ID NO: 17), whereasa reverse primer containing an XbaI restriction site, for example, and aregion complementary to the peptide(s) 3′ sequence.

The peptide-pAdTrack-CMV construct, confirmed by restriction digestionand DNA sequencing, can then be linerarized with PmeI and co-transformedwith the pAdEasy-1 adenoviral backbone into E. coli BJ5183 and selectedfor kanamycin resistance. The resulting recombinant plasmid can then betransfected into the 293 packaging cell line and the resultantAd-peptide adenoviral construct, e.g., an Ad5 serotype deleted for E1and E3, or a related adeno-associated virus, can then be amplified andpurified using CsCl density gradient centrifugation. Adenoviral titercan then determined by using standard assays before use in vitro or invivo. In this way, regulated expression of peptide(s) can be deliveredon demand and in situ (e.g., a wound bed with a chronic inability toheal wounds because of failed angiogenesis).

Anti-Angiogenic Compounds

Another embodiment is directed to compounds, compositions, and methodsfor inhibiting angiogenesis, tumor growth, ocular disease or any otherdisease or condition that is characterized or associated with abnormalangiogenesis or pathologic neovascularization. Although, as describedabove, angiogenesis is important to make new blood vessels, for example,to heal wounds and repair damage, as is known, pathologic angiogenesisrepresents a critical step in tumor progression through which the tumordevelops an autonomous blood supply, thus facilitating tumor growth.See, for example, Hanahan et al., Cell 100:57-70 (2000); Bergers et al.,Nat. Rev. Cancer 3:401-410 (2003). However, the creation of very smallblood vessels give a tumor its blood supply and allow it to be viableand grow. Anti-angiogenesis treatment is, therefore, a useful means fortargeting tumors and cutting off their blood supply, without whichtumors cannot grow. Without being bound by theory, these anti-angiogeniccompounds may or may not target the cancer cells and cancer stem cellsspecifically, but it is more likely that they target the blood vesselssupplying the cancer cells. In addition, other diseases that arecharacterized by unregulated blood vessel development including but notlimited to, for example, ocular diseases (e.g., macular degeneration anddiabetic retinopathy) are dependent upon angiogenesis orneovascularization, and may also benefit from the anti-angiogenesistherapy described herein.

Further, the methods of the present disclosure provide anti-angiogeniccompounds that inhibit the formation of new blood vessels (angiogenesisor neovascularization) required to establish and sustain tumors or thevascular complications of visually blinding disorders as is seen inaging and diabetes. The present invention additionally provides methodsand compositions that directly inhibit tumor growth, ocular disease, andother diseases or conditions associated with abnormal angiogenesis orneovascularization.

Diseases, disorders, or conditions associated with abnormalneovascularization or angiogenesis include, but are not limited to,retinal neovascularization, hemagiomas, solid tumor growth (includingsarcomas, lymphomas and carcinomas), metastasis, neovascular glaucoma,diabetic retinopathy, endometriosis, macular degeneration andretinopathy of prematurity (ROP). As used herein, the term“neovascularization” refers to the growth of blood vessels andcapillaries, and “angiogenesis,” as discussed above, refers to thephysiological process by which new blood vessels develop frompre-existing vessels. Normal angiogenesis is physiologic; abnormalangiogenesis is pathologic.

Through the inhibition of angiogenesis, one can intervene in theprogression or development of disease, ameliorate symptoms, or curedisease. Where the production of new blood vessels is required for thecontinued growth of abnormal tissue (e.g., cancer tissue), inhibitingangiogenesis will impede the blood supply to the abnormal tissue and,thus, reduce tissue mass due to lack of nutrients and oxygen beingdelivered to the abnormal tissue. Examples include cancerous tumors,proliferative diabetic retinopathy, inflammatory diseases, retinosis,macular degeneration, or any abnormally vascularized disease, conditionor disorder, among others. The methods of the present disclosure areeffective in part because the therapy is selective for angiogenesis.

Another embodiment of the disclosure is directed to anti-angiogeniccompounds that hybridize, recognize or bind to SEQ ID NOs: 5-16. Theseanti-angiogenic compounds may successfully inhibit the pathologicangiogenesis accompanying tumor growth when delivered systemically orthe neovascularization that takes place as a vision-threateningcomplication of diabetic retinopathy or age-related macular degenerationwhen delivered locally into the vitreal or intra-retinal compartment. Asdiscussed above, the present disclosure of peptides and nucleic acidsfor the purpose of wound healing promotes angiogenesis. Accordingly, byinhibiting angiogenesis, antisense oligonucleotides act in the reverseof the wound healing actions of the peptides.

The peptides of the disclosure also include peptides that arebiologically and/or immunologically active. The activity of thesepeptides can refer to a biological function (either inhibitory orstimulatory), such as but not limited to the promotion of cell migrationto a wound and cell proliferation of the cells that lead to healing of awound or heal a wound. The peptides of the disclosure may be used fordiagnostic purposes. For example, the peptides may be used to detectangiogenesis. Example 2 further exemplifies such a diagnostic use.

Just as the peptides of the present invention (SEQ ID NO: 1 and SEQ IDNO: 3) have been shown to induce angiogenesis in vitro and promotekeratinocyte migrations in response to injury in vitro, immunizingrabbits with SEQ ID NO: 1 and SEQ ID NO: 3 also elicits an immuneresponse generating polyclonal anti-angiogenic compounds.

Antisense Oligonucleotide Sequences

Another embodiment of the instant disclosure includes a nucleic acidsequence which hybridizes with a DNA or nucleotide sequence of SEQ IDNOs: 5-16, the complement thereof, or the cDNA under stringentconditions. These antisense oligonucleotides or nucleic acid sequencesthat are purified and isolated sequences are useful in inhibitingabnormal angiogenesis or neovascularization. The hybridizingpolynucleotides have at least about 70% sequence identity; at leastabout 80% identity; at least about 90%; or at least about 95% identitywith the polynucleotide of the present disclosure to which theyhybridize, where sequence identity is determined by comparing thesequences of the hybridizing polynucleotides when aligned so as tomaximize overlap and identity while minimizing sequence gaps. Themethods of determining percent identity are commonly known in the art.

Stringent hybridization conditions refers to an overnight incubation at42° C. in a solution comprising 50% formamide, 5×SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 micrograms/ml denatured, shearedsalmon sperm DNA, followed by washing the filters in 0.1×SSC at about65° C.

These antisense oligonucleotides or nucleic acid molecules or sequencesare also contemplated to hybridize to the polynucleotides of the presentdisclosure under lower stringency hybridization conditions. Bymanipulating the formamide concentration (lower percentages of formamideresult in lowered stringency); salt conditions, or temperature, thestringency of hybridization and signal detection may be altered. Forexample, lower stringency conditions include incubating overnight at 37°C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄; 0.02MEDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon sperm blockingDNA; followed by washing at 50° C. with 1×SSPE, 0.1% SDS. The ordinarilyskilled artisan understands that to achieve even lower stringency,multiple washes can be performed at higher salt concentrations (e.g.5×SSC) following stringent hybridization.

Variations in the above conditions may be accomplished by adding orsubstituting alternate blocking reagents used for suppressing theundesirable background in hybridization experiments. Typical blockingreagents include, but are not limited to, Denhardt's reagent, BLOTTO,heparin, denatured salmon sperm DNA, and commercially availableproprietary formulations. As understood in the art, the hybridizationconditions described above may require modification of certain blockingreagents due compatibility issues.

Thus, the present disclosure includes purified or isolated DNA sequencesencoding any of the peptides of SEQ ID NOs: 1-4, the DNA sequence beingselected from the group consisting of (a) the DNA sequences set out inSEQ ID NOs: 5-16 or their complementary strands; and (b) nucleic acidsequences which hybridize under stringent conditions to the DNAsequences defined in SEQ ID NOs: 5-16. The purified or isolated DNAsequence may consist essentially of a DNA sequence encoding apolypeptide having an amino acid sequence sufficiently duplicative ofthat of any of the peptides of SEQ ID NOs: 1-4 to allow possession ofthe biological property of causing angiogenesis tube formation, cellmigration or proliferation, and/or keratinocyte migration orproliferation. Further, the nucleic acid will be a functional peptideselected from SEQ ID NOs: 1-4. The DNA sequence may be a genomic DNAsequence, but will preferably be a cDNA sequence.

It has been shown that the effectiveness of these nucleic acid sequencesthat hybridize to the polynucleotide sequences of interest or antisensesequences may be beneficially modified to increase their stability invivo through modification of the backbone (including 2′-MOE(2′-O-2-methyloxy(ethyl)) modification, creating a phosphorothioatebackbone (replacing the non-binding phosphoryl oxygen atom with a sulferatom), or methylation of amino acids, such as but not limited tocytosines and uracils. See, for example, Gleave et al., U.S. Pat. No.6,900,187; Bennett et al., U.S. Pat. No. 6,111,094; Monia et al., J.Biol. Chem. 268:14514-14522 (1993); Miyake et al., Clin. Cancer Res.6:1655-1663 (2000); Miyake H et al., Expert Opinion on InvestigationDrugs 15:507-517 (2006); Monia et al., J. Biol. Chem. 268:14514-14522(1993); and Zellweger et al., J. Pharm. And Exp'l Therapeutics298:934-940 (2001).

Applications

In one embodiment, the wound healing molecules and compositions of thepresent disclosure can promote stimulation and growth of tissuesincluding epithelial tissue, which further includes simple or stratifiedsquamous, cuboidal and columnar epithelial tissue; connective tissuesuch as loose or dense, cartilage, adipose, bone, and blood connectivetissue (e.g., angiogenesis which occurs during wound healing); can beused for promoting stimulation and growth of muscle tissue such asvoluntary and involuntary, striated and smooth, and cardiac muscletissue; and nervous tissue such as central nervous system (CNS) tissue,which is comprised of the brain and spinal cord, and the peripheralnervous system (PNS) tissue, which is comprised of all the other nervoustissue in the body. The molecules and composition may also be used fororgan regeneration, reducing scarring, for cosmetic applications, suchas, cosmetic surgery, treating sun-damaged skin, wrinkles, promotinghair growth, as a haemostatic agent, or as a medium for growth of cellsand cultures. Types of wounds that may be treated include partial andfull-thickness wounds; pressure ulcers; venous ulcers; chronic vascularulcers; diabetic ulcers; civilian or military trauma wounds (abrasions,lacerations, second-degree burns, skin tears); drainage wounds andsurgical wounds (donor sites/grafts, post-Mohs' surgery, post-lasersurgery, podiatric, wound dehiscence). More particularly, the moleculesand compositions of the present invention can promote keratinocytemigration to a wound and enhance endothelial cell formation.

As used herein, the term “composition” is intended to mean at least oneor a plurality of components, elements or molecules of theaforementioned types, such as but not limited to, the wound healingpeptides or polypeptides, the polynucleotides encoding the wound healingpeptides or polypeptides, the oligonucleotides that hybridize to or arecomplementary to the polynucleotides of the instant disclosure, and thelike, directed against the wound healing peptide or polypeptides of thedisclosure, and the like with a carrier, vehicle, or diluent. Thecompositions of the disclosure may optionally contain growth factors inisolated and/or purified form, as well as synthetic growth factors.Non-limiting examples of useful growth factors in the compositioninclude one or more of any of the following: PDGF-AA, PDGF-BB, PDGF-AB,EGF, VEGF, TGF-alpha, FGF, TGF-beta, IGF-1, IGF-2, NGF, anderythropoietin, and/or cytokines generally, and/or lymphokinesgenerally, and/or interleukins, and/or monokines As used herein (anddescribed below), percentages are based on the weight of thecomposition.

Preferably, the composition will comprise of or consist essentially ofonly components that do not alter the basic novel characteristics ofpromoting cell migration to a wound and cell proliferation of the cellsthat lead to healing of a wound and/or heal a wound. The disclosedcomposition may therefore comprise or consist essentially of one or moreof the foregoing molecules and/or factors. However, one embodiment ofthe disclosure is directed to a composition that excludes componentshaving de minimus, or are nonessential, or no effect on the basic andnovel characteristics of the wound healing ability of the components ofthe composition. In another embodiment, the disclosed compositionsexclude components that have de minimus, or are nonessential, or noeffect on the basic and novel characteristics of the anti-angiogenesischaracteristics of those particular components useful for such purposes.

When a composition includes growth factors, in terms of nanograms permilliliters, the growth factors may be included in an amount of fromabout 10 ng/ml to about 500 ng/ml growth factors, more preferably fromabout 40 ng/ml to about 300 ng/ml growth factors, and most preferablyfrom about 90 ng/ml to about 220 ng/ml growth factors. Preferably, thegrowth factors will be platelet derived growth factors (PDGF) orvascular endothelial growth factor (VEGF).

Further examples of wound healing and/or repairing and/or growthpromoting agents that may be combined with or used in combination withthe compounds according to the present disclosure may include porcinederived agents such as OASIS™ manufactured by Cook Biotech Incorporatedand distributed by Healthpoint, Ltd., San Antonio, Tex.; or one or morecarriers or vehicles, e.g., purified water, glycerin, carboxymethylcellulose, sodium, allantoin, benzyl alcohol, methylparaben,propylparaben, each of which is found in SoloSite™, by Smith and Nephew,Largo, Fla. The wound healing compounds may also be used in conjunctionwith scaffolds to assist in the angiogenesis and wound healing.

In embodiments wherein one or more additional growth promoting activeagents, one or more antibacterial agents, one or more pain relievers,one or more vitamins, one or more minerals, and/or one or more herbalfactors are included, the composition will include an effective amountof the one or more of the molecules according to the invention, aneffective amount of the one or more additional growth promoting activeagents, an effective amount of the one or more antibacterial agents, aneffective amount of the one or more pain relievers, an effective amountof the one or more vitamins, an effective amount of the one or moreminerals, and an effective amount of the one or more herbal additives.

Examples of suitable carriers and/or vehicles, such as pharmaceuticallyacceptable carriers, include one or more of collagen, such asmicrocrystalline collagen, creams, microcapsules, oils, aloe vera, awax, a polyol, one or more fats or oils, one or more emulsifying agents,and/or one or more water-soluble gums, water, saline, stearyl alcoholNF, white petrolatum USP, polyoxyl 40, stearate NF, carboxymethylcellulose, lanolin, alginate, such as calcium alginate, gel, propyleneglycol USP, isopropyl myristate NF, and/or sorbitan monooleate NF with0.3% methylparaben NF. The composition may optionally include apreservative. The carrier and/or vehicle may be included in thecomposition in an amount from about 1% to about 99% of the composition,preferably from about 25% to about 50%, most preferably from about 30%to about 40% of the total composition or combination.

Examples of suitable additional growth promoting active agents includeepidermal growth factors, steroids, enzymes, and hormones, natural (suchas having been isolated and purified) or synthetic. The additionalgrowth promoting active agents may be included in the composition in anamount of from about 1% to about 50% of the composition.

Examples of suitable antibacterial agents that may be applied before,during, or after treatment with the composition as a solution or acream, gel, or a paste, include silver compounds, such as silvernitrate, honey, sulfamylon, silver sulfadiazine, such as a micronizedsilver sulfadiazine cream (e.g., THERMAZENE™ by Kendall, Mansfield,Mass.), saline; neosporin, and/or a mycin, such as vancomycin,gintamycin, erythromycin or derivative, and/or a cillin, such as apenicillin, or amoxicillin. Other antimicrobial agents include iodine,such as beads of cadexomer iodine found in IODOSORB™ GEL, byHealthpoint™, San Antonio, Tex. The antibacterial agents may be includedin the composition in an amount of from about 1% to about 25% of thecomposition.

The compounds and compositions of the present invention can also becombined with commercially available wound repairing or healingdressings, such as, for example, a sodium chloride dressing (e.g.,Mesalt™ by Molnlycke Health Care A B, Goteborg, Sweden), a silverantimicrobial dressing (e.g., SilvaSorb™ by AcryMed, Inc., Portland,Oreg. and Acticoat* or Acticoat*7 by Smith & Nephew, Inc., Largo, Fla.),a silver impregnated antimicrobial dressing (e.g., Aquacel™ by ConvaTecLimited, Division of E. R. Squibb and Sons, Inc., Princeton, N.J. andMaxorb™ by Medline Industries, Inc., Mundelein, Ill.), a sodium alginatesilver oxide dressing, optionally containing sustained-release polymersthat dissolve in water releasing silver ions into the wound (e.g.,Argiaes™ Powder by Medline Industries, Inc., Mundelein, Ill.), ahydrocolloid dressing, optionally containing an inner wound contactlayer of hydrocolloids contained within an adhesive polymer matrix andan outer layer of polyurethane film (e.g., SignaDress™ DuoDerm™ byConvaTec Limited, Division of E. R. Squibb and Sons, Inc., Princeton;N.J.), a collagen and/or calcium alginate dressing (e.g., Fibracol™ byJohnson and Johnson Medical, Skipton, United Kingdom and AlgiSite* M bySmith & Nephew, Inc., Largo, Fla.), a dressing layer containing softsilicone (e.g., Mepitel™ by Molnlycke Health Care A B, Goteborg,Sweden), a dressing containing polyhexamethylene biguanide and/orcellulose (e.g., XCell™ by XYLOS Corporation, Langhorne, Pa.), adressing containing hyaluronic acid or an ester of hyaluronic acid(e.g., Hyaff™, Hyalofill™ F, or Hyalofill™ R by ConvaTec Limited,Division of E. R. Squibb and Sons, Inc., Princeton, N.J.), a dressingmade of sponge, optionally containing hydrofera bacteriostatic polyvinylalcohol sponge (e.g., Hydrofera Blue™ by Hydrofera™, Willimantic,Conn.), and/or a dressing or pad containing spherical hydrophilic beadsof cadexomer, optionally containing iodine and/or polyethylene glycol(e.g., Iodoflex™ Pad by Healthpoint, Ltd., San Antonio, Tex.).

The compounds and compositions of the present invention can further becombined with commercially available wound repairing or healingointments, such as, for example, an ointment containing papain, which isderived from papaya (e.g., Panafil™ or Accuzyme™ by Healthpoint, Ltd.,Forth Worth, Tex.).

The compounds and compositions of the present invention can also becombined with commercially available wound repairing or healing gels,such as, for example, a sodium chloride gel (e.g., Hypergel™ byMolnlycke Health Care A B, Goteborg, Sweden); and/or gels containing oneor more of the following ingredients water, glycerin, glycereth-7,polyvinylpyrrolidone, carbomer, triethanolamine, EDTA, propylene glycol,diazolidinyl urea, methylparaben, and propylparaben, such as foundtogether in 3M™ Tegagel™ Hydrogel Wound Filler by 3M Heath Care, St.Paul, Minn.

The compounds and compositions of the present invention can further becombined with commercially available wound repairing or healing sprays,such as, for example, a spray containing papain (e.g., Panafil™ Spray byHealthpoint, Ltd., Forth Worth, Tex.).

The compounds and compositions of the present invention can further becombined with commercially available wound repairing or healingemulsions, such as, for example, a water-based emulsion, optionallycontaining one or more of the following ingredients liquid paraffin,ethylene glycol monostearate, stearic acid, propylene glycol, paraffinwax, squalane, avocado oil, trolamine/sodium alginate, triethanolamine,cetyl palmitate, methylparaben (sodium salt), sorbic acid (as potassiumsalt), propylparaben (sodium salt), and/or fragrances (e.g., Biafine™ byMedix Pharmaceuticals Americas, Inc., Largo, Fla.).

Examples of suitable pain relievers and anti inflammatory agents includeheparin, bromelain, ozone, analgesics, opioids, and acetaminophen. Thepain relievers and anti-inflammatory agents may be included in thecomposition in an amount of from about 1% to about 25% of thecomposition depending on the type of wound and possibility of infection.

Examples of vitamin factors that may be used in the compositions of theinvention or in combination therapy include Vitamin A and/or retinoids,Vitamin E, Vitamin C, Vitamin D, folic acid, vitamin B5, Bromelain,Vitamin B-complex, Zinc (oral and topical), Chondroitin sulfate(topical), Copper, Ornithine alpha-ketoglutarate (OKG), Arginine,Carnosine, chondroitin sulfate (oral), Glucosamine sulfate (oral),icthammol, calamine, silver sulphadiazine, chlorohexadine acetate, coal,tar. The vitamin factors may be included in the composition in an amountof from about 0.1% to about 25% of the composition.

Examples of minerals that may be used in the compositions of theinvention include copper, magnesium, manganese, zinc, iron. The mineralfactors may be included in the composition in an amount of from about0.1% to about 25% of the composition.

The compounds and compositions will generally be stored in a container,such as a sealed container, or a water resistant sealed container.

Examples of herbal factors that may be used in the compositions of theinvention include Aloe vera (topical), Chamomile (topical), Gotu kola(oral and topical), Honey (topical), Horse chestnut (topical), Arnica(topical), Bladderwrack (topical), Calendula (topical), Chaparral(topical), Comfrey (topical), Echinacea (topical), Horsetail (oral andtopical), Plantain (topical), St. John's wart (topical), Tea tree oil(topical), goldenseal (topical), echinacea (topical), and Witch hazel(topical). The herbal factors may be included in the composition in anamount of from about 1 mg to about 6 mg and make up from about 0.1% toabout 25% of the composition.

In another embodiment, anti-angiogenic compounds can be used for testingfor pathologic angiogenesis and/or effectiveness of cancer drugcandidate compounds or compositions for treating forms of cancer and/orallied cancer diseases. These anti-angiogenic compounds mayadvantageously be used to treat various types of vascularized cancersthat progress and metastasize, including sarcomas (e.g., breast cancer),lymphomas and carcinomas (e.g., lung cancer), among other types ofvascular tumor types. A further embodiment relates to the use ofanti-angiogenic compounds for treating various types of ocular diseases(i.e., vision altering complications observed during diabeticretinopathy, choroidal neovascularization or age-related maculardegeneration; ocular vascular proliferative lesions accompanyingdiabetes (diabetic retinopathy), choroidal neovascularization (CNV) orage-related macular degeneration (AMD)).

The present disclosure contains applications which may utilize both thepeptides of the present disclosure and the anti-angiogenic compounds ofthe present disclosure. For example, a person may require administrationof anti-angiogenic compounds to combat pancreatic cancer orglioblastoma, while requiring an intravitreal injection of humanizedanti-angiogenic compound for treatment of wet AMD or diabeticretinopathy. At the same time, a diabetic patient may need localapplication of trace peptide levels for local promotion of wound healingangiogenesis and activation of non-healing plantar ulcer into one thatcan heal and progress to wound closure. Upon completion of treatment,subsequently require administration of any combination of the peptidesdisclosed herein to reduce scarring.

Administration

A further embodiment of the present disclosure provides for theadministration of, for example, the wound healing peptides, to a subjectin need, by any suitable route including but not limited to, topicallyor by parenteral means, including subcutaneous and intramuscularinjection, implantation of sustained release depots, intradermalinjection, and the like. Any local administration is most useful for theembodiments of the disclosure. Topical formulations comprising the woundhealing peptides include creams, ointments, aerosols, bandages and otheradvanced wound dressings, and the like.

Formulations of the present invention may be any that are appropriatefor the route of administration and will be apparent to those skilled inthe art. Accordingly, the composition of the present disclosure having apharmaceutically- and/or physiologically-acceptable carrier, vehicle, ordiluent. Such compositions of the present invention may be aqueoussolutions, creams, emulsions, ointments, suspensions, gels, liposomalsuspensions, and the like. Pharmaceutically acceptable carriers are wellknown in the art such as saline, and may also comprise bulking agents,other medicinal preparations, adjuvants and any other suitablepharmaceutical ingredients. Aqueous carriers can contain more than onebuffer salt, as well as salts such as sodium and potassium chlorides,dextrose, polyethylene glycol and other solutes. One of skill in the artmay also include suitable preservatives, antioxidants, stabilizers,antimicrobials, and buffering agents, for example, BHA, BHT, citricacid, ascorbic acid, and tetracycline.

Further, one may provide a peptide or anti-angiogenic compound of thepresent disclosure in solid form, particularly as a lyophilized powder.Lyophilized formulations generally contain stabilizing and bulkingagents, such as human serum albumin, sucrose, and mannitol. As theskilled artisan will appreciate, the lyophilized powders may bereconstituted with any of the aforementioned diluents, carriers, andvehicles and used in the appropriate manner. The anti-angiogeniccompound of the disclosure may be formed in a lyophilized solid forlong-term storage, ease of use or transport, and the like. For example,when the lyophilized solid comprises the wound healing peptides of thedisclosure, the lyophilized solid may be used under combat conditions asa solid for direct application to open wounds (i.e., sprinkle the powderinto a wound). Another embodiment is directed to a reconstitutedlyophilized powder comprising the anti-angiogenic compound that inhibitsor reduces vascularization of cancer cells such as those found intumors, where the lyophilized powder is reconstituted with a diluent forlocal injection.

The term “effective amount” refers to an amount of peptide or antisenseoligonucleotide sufficient to exhibit a detectable therapeutic effect.The therapeutic effect may include, for example, without limitation,promoting the growth of tissue or cells curing the process of woundhealing, promoting angiogenesis (neovascularization) in wound healing,inhibiting angiogenesis in cancerous cells, inhibiting angiogenesis incancerous cells, tumors or macular degeneration, inhibition of tumorcell growth, and the like. The precise effective amount foradministration to a subject will depend upon the subject's size andhealth, the nature and severity of the condition to be treated, and thelike. The effective amount for a particular individual can be determinedby routine experimentation based on the information provided herein andas calculated by the skilled practitioner based on dose response assaysand animal models that correlate to human applications.

The term “pharmaceutically acceptable” refers to compounds,compositions, and carriers, vehicles, and diluents, which may beadministered to mammals, including humans, without undue toxicity.

With regard to topical applications, peptides of the present disclosurewill be utilized in effective amounts for the promotion of angiogenesis.For example, such effective amounts range from about subnanomolar toabout micromolar, preferably from about 0.5 nM to about 250 nM, and mostadvantageously from about 10 nM to about 100 nM. The effective amountsrange from about 6×10⁻¹⁰% to about 6×10⁻⁶% of the weight of the subject.For internal use, the formulation may be released directly into theregion to be treated either from implanted slow release polymericmaterial or from slow release pumps or repeated injections. The releaserate in either case is about 100 ng to about 100 mg/day/cm³.

Systemic dosages of anti-angiogenic compounds generally depend on theage, weight and conditions of the patient and on the administrationroute.

The content of all patents, patent applications, published articles,abstracts, books, reference manuals and abstracts, as cited herein arehereby incorporated by reference in their entireties to more fullydescribe the state of the art to which the disclosure pertains.

It should be understood that the foregoing description is onlyillustrative of the present disclosure. Various alternatives andmodifications can be devised by those skilled in the art withoutdeparting from the disclosure. Accordingly, the present disclosure isintended to embrace all such alternatives, modifications and variationsthat fall within the scope of the appended claims.

EXAMPLES

The following Examples further describe and demonstrate embodimentswithin the scope of the present disclosure. The examples are givensolely for the purpose of illustration and are not to be constructed aslimitations of the present disclosure, as many variations thereof arepossible without departing from the spirit and scope of the disclosure.

Example 1

Crude raw lyophilized pooled human-platelet rich plasma, prepared inaccordance with the procedures disclosed in Gandy U.S. PatentPublication No. 20060142198 and Gandy U.S. Patent Publication No.20060004189, was obtained. The crude material was treated, biochemicallyseparated, and enriched by size exclusion and ion exchangechromatography DEA Cellulose to produce a fraction having enrichedactivity. Tandem Mass Spectrometry (MS/MS) combined with databasesearching was employed to identify the components contained in a sampleof the raw material. Two unnamed peptide sequences, SEQ ID NO: 1 and SEQID NO: 2, were identified and selected for investigation.

The peptides identified were then synthesized at the Tufts UniversityCore Facility using an ABI 431 Peptide Synthesizer employing FastMocChemistry. The isolated and purified peptides were then re-characterizedby Mass Spectrometry analysis and analytical HPLC chromatography. Thetwo peptide sequences, SEQ ID NO: 1 and SEQ ID NO: 2, were then combinedto form a new peptide, i.e., SEQ ID NO: 3.

Example 2

Each of the three peptide sequences were then analyzed for activity via(1) in vitro angiogenesis assays where blood vessel formation in vitrowas quantified; (2) in vitro response to injury analyses, includingquantitative analysis, of migration in response to injury; and (3) invitro quantitation of cell proliferation assays. The results ofexperiments performed are illustrated in FIGS. 1 to 4.

The peptide (SEQ. ID NO: 1 (“UN1”)) was compared to each of VEGF 4 ng/mland Serum Control in an in vitro angiogenesis analysis. As illustratedin FIGS. 1 and 2, the peptide of the invention induced significanttubule formation and process length in comparison to the Serum Controland VEGF.

The peptide (SEQ ID NO: 3 (“UN3”)) was also compared to VEGF and SerumControl in in vitro angiogenesis analysis. As illustrated in FIG. 3, thepeptide of the invention showed a significant increase in process lengthin comparison with Serum Control. It also showed a significant increasein process length in comparison to VEGF at an amount of 250 nM.

In addition, each of the three peptides of the invention were comparedwith regard to adult normal human epithelial keratinocyte migration. Asillustrated in FIG. 4, each performed substantially better than thecontrol.

1-23. (canceled) 24) A method of treating a subject with a wound,comprising administering to the subject in need, an effective amount ofone or more than one isolated peptide selected from the group consistingof: a) an isolated peptide comprising SEQ ID NO: 1; b) an isolatedpeptide comprising SEQ ID NO: 2; c) an isolated peptide encoded by apolynucleotide comprising SEQ ID NO: 5; and d) an isolated peptideencoded by a polynucleotide comprising SEQ ID NO:
 9. 25) The method ofclaim 24, wherein the one or more than one isolated peptide isadministered in a composition further comprising a carrier, vehicle, ordiluent. 26) The method of claim 25, wherein the composition is topical.27) The method of claim 24, wherein the wound is a wound selected fromthe group consisting of: acute accidental injury, surgical acuteinjuries, chronic wounds, ulcers, thermal injuries, combat casualtyinjuries, hypertrophic scarring, and any combinations thereof. 28) Amethod of treating a subject with abnormal vascularization, comprisingadministering to a subject in need, an effective amount of one or morethan one isolated polynucleotide selected from the group consisting of:a) an isolated polynucleotide fully complementary to an isolatedpolynucleotide encoding a peptide comprising SEQ ID NO: 1; b) anisolated polynucleotide fully complementary to an isolatedpolynucleotide encoding a peptide comprising SEQ ID NO: 2; c) anisolated polynucleotide fully complementary to an isolatedpolynucleotide comprising SEQ ID NO: 5; and d) an isolatedpolynucleotide fully complementary to an isolated polynucleotidecomprising SEQ ID NO:
 9. 29) The method of claim 26, wherein the one ormore than one isolated polynucleotide is administered in a compositionfurther comprising a carrier, vehicle, or diluent. 30) The method ofclaim 26, wherein abnormal vascularization is a disease or conditionselected from the group consisting of: vascular tumors, sarcomas,carcinomas, lymphomas, ocular diseases, macular degeneration, diabeticretinopathy, age-related macular degeneration, and any combinationsthereof. 31) A method of treating a subject with abnormalvascularization, comprising administering to a subject in need, aneffective amount of an anti-angiogenic compound comprising an isolatedpolynucleotide fully complementary to an isolated polynucleotidecomprising one or more polynucleotide sequences selected from the groupconsisting of SEQ ID NOs: 5-12. 32) The method of claim 31, whereinabnormal vascularization is a disease or condition selected from thegroup consisting of: vascular tumors, sarcomas, carcinomas, lymphomas,ocular diseases, macular degeneration, diabetic retinopathy, age-relatedmacular degeneration, and any combinations thereof. 33) A method ofinhibiting vascularization, comprising administering a compoundcomprising an isolated polynucleotide fully complementary to an isolatedpolynucleotide comprising one or more polynucleotide sequences of claim31.